P1593: DECIPHERING UNIQUE HETEROGENEITY AND EFFICACY DIFFERENCES IN IMMUNE THROMBOCYTOPENIA AT THE SINGLE-CELL LEVEL
Authors: Min Xu, Lu Tang, Yu Hu, Heng Mei
Journal: HemaSphere (2023)

Topic: 32. Platelet disorders Background: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by decreased platelet counts and increased bleeding risk. Although most patients respond to treatment of corticosteroids and TPO receptor agonists, some patients remain unresponsive and prone to develop chronic or refractory/relapsed disease. Aims: The purpose of this study was to reveal the unique immune heterogeneity of ITP and the differences between responders and non-responders at the single-cell level. Methods: Here, we used the Rhapsody™ single-cell analysis system to label specific cell tags and demultiplex full-length cDNA molecules generated by the BD Rhapsody platform from peripheral blood mononuclear cells (PBMCs) to identify subsets of immune cells that contribute to the pathogenesis and curative effect difference of ITP. The flow cytometry had been performed to further define the immune cell subsets on PBMCs. Results: Using single-cell RNA-seq, we profiled ~138755 PBMCs from 24 ITP patients and 4 matched controls. In general, increased expression of megakaryocyte cells, myeloid cells and peripheral blood stem cell (PBSC) distinguished ITP patients from healthy control. Elevated myeloid cells, B cells, megakaryocytes and decreased CD4+ T cells observed in non-responding patients compared with responding patients. Subgroup analyzes showed that myeloid cells and B cells were increased and CD4+ T cells were decreased in corticosteroids-unresponsive patients, while myeloid cells were decreased and CD4+T cells were increased in TPO-RAs-unresponsive patients. In addition, with the prolongation of disease course, an increase in myeloid cells and a decrease in CD4+ T cells were observed. And GSVA enrichment analysis revealed that signaling pathway changes mainly focused on platelet function, megakaryocyte production and differentiation, and complement activation. Then, cluster analysis of B cells found that B_naive_3 cluster was significantly elevated in ITP patients, accompanied by the higher expression of molecular markers like JUNB, FOS, FOSB, IL-32, CCL5, which may be the main cell groups and biomarkers that producing autoantibodies in ITP patients. Finally, subcluster analysis of T cells revealed elevated CD4+/CD8+ cytotoxic T cells and decreased CD4+/CD8+ naïve T cells and Tregs in ITP patients compared with control. And corticosteroids sensitive patients have an increased Fos-Jun-CD4+ T cells, TPO-RAs sensitive patients have an increased Fos-Jun-CD4+ T cells, Fos-Jun-NK cells and a decreased STAT1-CD4+T cells. Interestingly, both Fos-Jun-CD4+ T cells and Fos-Jun-NK cells were decreased with the prolongation of the disease. Summary/Conclusion: Our data provide evidence of the heterogeneity of cellular and molecular pathways involved in the pathogenesis of ITP and reveal the factors affecting the efficacy of the most important drug corticosteroids and TPO receptor agonists from the single-cell level, which lays the groundwork for precision medicine applications of immune thrombocytopenia.Fig: A. Clustering display of tsne after clustering analysis of all PBMCs. B. The difference in cell ratio between ITP patients and controls in general; C. Cluster analysis of B cells; D. The difference in B cells subsets between ITP patients and controls; E. The difference in T cells subsets between ITP patients and controls, and curative effect difference of patients use corticosteroids and TPO receptor agonists Keywords: Thrombopoietin (TPO), RNA-seq, Immune thrombocytopenia (ITP), T cell